Detection of nucleic acids by type specific hybrid capture method

ABSTRACT

Target-specific hybrid capture (TSHC) provides a nucleic acid detection method that is not only rapid and sensitive, but is also highly specific and capable of discriminating highly homologous nucleic acid target sequences. The method produces DNA-RNA hybrids which can be detected by a variety of methods.

FIELD OF INVENTION

This invention relates to the field of nucleic acid detection methods ingeneral and more particularly relates to the detection of nucleic acidsby target-specific hybrid capture method.

BACKGROUND OF THE INVENTION

The detection of specific nucleic acid sequences present in a biologicalsample is important for identifying and classifying microorganisms,diagnosing infectious diseases, detecting and characterizing geneticabnormalities, identifying genetic changes associated with cancer,studying genetic susceptibility to disease, and measuring response tovarious types of treatment. A common technique for detecting andquantitating specific nucleic acid sequences is nucleic acidhybridization.

Various hybridization methods are available for the detection and studyof nucleic acids. In a traditional hybridization method, the nucleicacids to be identified are either in a solution or affixed to a solidcarrier. The nucleic acids are detected using labelled nucleic acidprobes which are capable of hybridizing to the nucleic acids. Recently,new hybridization methods have been developed to increase thesensitivity and specificity of detection. One example is the hybridcapture method described in U.S. application Ser. No. 07/792,585.Although these new hybridization methods offer significant improvementsover the traditional methods, they still lack the ability to fullydiscriminate between highly homologous nucleic acid sequences.

It is therefore an object of the present invention to provide ahybridization method which is not only rapid and sensitive, but is alsohighly specific and capable of discriminating highly homologous nucleicacid target sequences.

SUMMARY OF THE INVENTION

The present invention provides a novel nucleic acid detection method,referred to herein as target-specific hybrid capture (“TSHC”). TSHC is ahighly specific and sensitive method which is capable of discriminatingand detecting highly homologous nucleic acid target sequences.

In one embodiment, the method relates to detecting a target nucleic acidwherein the targeted nucleic acid is hybridized simultaneously, orsequentially, to a capture sequence probe and an unlabelled signalsequence probe. These probes hybridize to non-overlapping regions of thetarget nucleic acid and not to each other so that double-strandedhybrids are formed. The hybrids are captured onto a solid phase anddetected. In a preferred embodiment, an DNA-RNA hybrid is formed betweenthe target nucleic acid and the signal sequence probe. Using thismethod, detection may be accomplished, for example, by binding a labeledantibody capable of recognizing an DNA-RNA hybrid to the double-strandedhybrid, thereby detecting the hybrid.

In another embodiment, the signal sequence probe used in the detectionmethod is a nucleic acid molecule which comprises a DNA-RNA duplex and asingle stranded nucleic acid sequence which is capable of hybridizing tothe target nucleic acid. Detection may be accomplished, for example, bybinding a labeled antibody capable of recognizing the DNA-RNA duplexportion of the signal sequence probe, thereby detecting the hybridformed between the target nucleic acid, the capture sequence probe andthe signal sequence probe.

In yet another embodiment, the signal sequence probe used in thedetection method is a molecule which does not contain sequences that arecapable of hybridizing to the target nucleic acid. Bridge probescomprising sequences that are capable of hybridizing to the targetnucleic acid as well as sequences that are capable of hybridizing to thesignal sequence probe are used. In this embodiment, the signal sequenceprobe comprises a DNA-RNA duplex portion and a single stranded DNAsequence portion containing sequences complementary to sequences withinthe bridge probe. The bridge probe, which hybridizes to both the targetnucleic acid and the signal sequence probe, therefore serves as anintermediate for connecting the signal sequence probe to the targetnucleic acid and the capture sequence probe hybridized to the targetnucleic acid.

In another embodiment of the TSHC method of the invention, blockerprobes comprising oligonucleotides complementary to the capture sequenceprobes are used in the method to eliminate excess capture sequenceprobe, thereby reducing the background signal in detection andincreasing specificity of the assay.

The present invention also relates to novel probes. These probes arenucleic acid sequences which can function in various hybridizationassays, including, for example, the TSHC assay.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a schematic diagram illustrating one embodiment of thetarget-specific hybrid capture method.

FIG. 2 is a schematic diagram illustrating one embodiment of thetarget-specific hybrid capture method.

FIG. 3 is a schematic diagram illustrating possible mechanisms of actionof an embodiment that employs fused capture sequence probes intarget-specific hybrid capture detection.

FIG. 4 shows the analytical sensitivity and specificity oftarget-specific hybrid capture detection of HSV-1.

FIG. 5 shows the analytical sensitivity and specificity oftarget-specific hybrid capture detection of HSV-2.

FIGS. 6A-6D show the various embodiments of the target-specific hybridcapture-plus method.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for detecting the presence ofnucleic acids in test samples. More specifically, the invention providesa highly specific and sensitive method which is capable ofdiscriminating and detecting highly homologous nucleic acid sequences.

Any source of nucleic acid, in purified or non-purified form, can beutilized as the test sample. For example, the test sample may be a foodor agricultural product, or a human or veterinary clinical specimen.Typically, the test sample is a biological fluid such as urine, blood,plasma, serum, sputum or the like. Alternatively the test sample may bea tissue specimen suspected of carrying a nucleic acid of interest. Thetarget nucleic acid in the test sample may be present initially as adiscrete molecule so that the sequence to be detected constitutes theentire nucleic acid, or may only be a component of a larger molecule. Itis not necessary that the nucleic acid sequence to be detected bepresent initially in a pure form. The test sample may contain a complexmixture of nucleic acids, of which the target nucleic acid maycorrespond to a gene of interest contained in total human genomic DNA orRNA or a portion of the nucleic acid sequence of a pathogenic organismwhich organism is a minor component of a clinical sample.

The target nucleic acid in a test sample can be DNA or RNA, such asmessenger RNA, from any source, including bacteria, yeast, viruses, andthe cells or tissues of higher organisms such as plants or animals.Methods for the extraction and/or purification of such nucleic acids arewell known in the art. Target nucleic acids may be double-stranded orsingle-stranded. In the present method, it is preferred that the targetnucleic acids are single-stranded or made single-stranded byconventional denaturation techniques prior to the hybridization steps ofthe method. In a preferred embodiment, base denaturation technique isused to denature the double-stranded target DNA.

The term “oligonucleotide” as the term is used herein refers to anucleic acid molecule comprised of two or more deoxyribonucleotides orribonucleotides. A desired oligonucleotide may be prepared by anysuitable method, such as purification from a naturally occurring nucleicacid, by molecular biological means, or by de novo synthesis. Examplesof oligonucleotides are nucleic acid probes described herein.

Nucleic acid probes are detectable nucleic acid sequences that hybridizeto complementary RNA or DNA sequences in a test sample. Detection of theprobe indicates the presence of a particular nucleic acid sequence inthe test sample. In one embodiment, the target-specific hybrid capturemethod employs two types of nucleic acid probes: capture sequence probe(CSP) and signal sequence probe (SSP). A capture sequence probecomprises a nucleic acid sequence which is capable of hybridizing tounique region(s) within a target nucleic acid and being captured onto asolid phase. A signal sequence probe comprises a nucleic acid sequencewhich is capable of hybridizing to regions within a target nucleic acidthat are adjacent to the unique regions recognized by the CSP. Thesequences of CSP and SSP are selected so that they would not hybridizeto the same region of a target nucleic acid or to each other.

In addition, the CSP and the SSP are selected to hybridize to regions ofthe target within 50,000 bases of each other. The distance between thesequence to which the CSP hybridizes within the target nucleic acid andthe sequence to which the SSP hybridizes is preferably between 1 to50,000 bases, more preferably, the distance is less than 3,000 bases.Most preferably, the distance is less than 1,000 bases.

The CSP used in the detection method can be DNA, RNA, peptide nucleicacids (PNAs) or other nucleic acid analogues. PNAs are oligonucleotidesin which the sugar-phosphate backbone is replaced with a polyamide or“pseudopeptide” backbone. In a preferred embodiment, the CSP is DNA. TheCSP has a minimum length of 8 bases, preferably between 15 to 100 baseslong, and more preferably between 20 to 40 bases long. The CSP issubstantially complementary to the sequence within a target nucleic acidto which it hybridizes. The sequence of a CSP is preferably at least 75%complementary to the target hybridization region, more preferably, 100%complementary to this sequence. It is also preferred that the CSPcontains less than or equal to 75% sequence identity, more preferablyless than 50% sequence identity, to non-desired sequences believed to bepresent in a test sample. The sequence within a target nucleic acid towhich a CSP binds is preferably 12 bases long, more preferably 20-40bases long. It may also be preferred that the sequences to which the CSPhybridizes are unique sequences or group-specific sequences.Group-specific sequences are multiple related sequences that formdiscrete groups.

In one embodiment, the CSP used in the detection method may contain oneor more modifications in the nucleic acid which allows specific captureof the probe onto a solid phase. For example, the CSP may be modified bytagging it with at least one ligand by methods well-known to thoseskilled in the art including, for example, nick-translation, chemical orphotochemical incorporation. In addition, the CSP may be tagged atmultiple positions with one or multiple types of labels. For example,the CSP may be tagged with biotin, which binds to streptavidin; ordigoxigenin, which binds to anti-digoxigenin; or 2,4-dinitrophenol(DNP), which binds to anti-DNP. Fluorogens can also be used to modifythe probes. Examples of fluorogens include fluorescein and derivatives,phycoerythrin, allo-phycocyanin, phycocyanin, rhodamine, Texas Red orother proprietary fluorogens. The fluorogens are generally attached bychemical modification and bind to a fluorogen-specific antibody, such asanti-fluorescein. It will be understood by those skilled in the art thatthe CSP can also be tagged by incorporation of a modified basecontaining any chemical group recognizable by specific antibodies. Othertags and methods of tagging nucleotide sequences for capture onto asolid phase coated with substrate are well known to those skilled in theart. A review of nucleic acid labels can be found in the article byLandegren, et al. “DNA Diagnostics-Molecular Techniques and Automation”,Science, 242:229-237 (1988), which is incorporated herein by reference.In one preferred embodiment, the CSP is tagged with biotin on both the5′ and the 3′ ends of the nucleotide sequence. In another embodiment,the CSP is not modified but is captured on a solid matrix by virtue ofsequences contained in the CSP capable of hybridization to the matrix.

The SSP used in the detection method may be a DNA or RNA. In oneparticular embodiment of the invention, the SSP and target nucleic acidform a DNA-RNA hybrid. Therefore, in this embodiment, if the targetnucleic acid is a DNA, then the preferred SSP is an RNA. Similarly, ifthe target nucleic acid is RNA, then the preferred SSP is a DNA. The SSPis generally at least 15 bases long. However, the SSP may be up to orgreater than 1000 bases long. Longer SSPs are preferred. The SSP maycomprise a single nucleic acid fragment, or multiple smaller nucleicacid fragments each of which is preferably between 15 to 100 bases inlength.

In another embodiment, the SSP used in the detection method comprises aDNA-RNA duplex and a single stranded nucleic acid sequence capable ofhybridizing to the target nucleic acid (FIG. 6A). The SSP may beprepared by first cloning a single stranded DNA sequence complementaryto sequences within the target nucleic acid into a single-stranded DNAvector, then hybridizing RNA complementary to the DNA vector sequence togenerate a DNA-RNA duplex. For example, if M13 is used as the DNAvector, M13 RNA is hybridized to the M13 DNA sequence in the vector togenerate a DNA-RNA duplex. The resulting SSP contains a DNA-RNA duplexportion as well as a single stranded portion capable of hybridizing tosequences within the target nucleic acid. The single stranded DNA shouldbe at least 10 bases long, and may be up to or greater than 1000 baseslong. Alternatively, the DNA-RNA duplex portion of the SSP may be formedduring or after the reaction in which the single stranded portion of theSSP is hybridized to the target nucleic acid. The SSP can be linear,circular, or a combination of two or more forms. The DNA-RNA duplexportion of the SSP provides amplified signals for the detection ofcaptured hybrids using anti-DNA-RNA antibodies as described herein.

In yet another embodiment, the SSP used in the detection method is amolecule which does not contain sequences that are capable ofhybridizing to the target nucleic acid. In this embodiment, bridgeprobes comprising sequences capable of hybridizing to the target nucleicacid as well as sequences capable of hybridizing to the SSP are used.The bridge probes can be DNA, RNA, peptide nucleic acids (PNAs) or othernucleic acid analogues. In one embodiment (FIG. 6B), the SSP comprises aDNA-RNA duplex portion and a single stranded portion containingsequences complementary to sequences within the bridge probe. The bridgeprobe, which is capable of hybridizing to both the target nucleic acidand the SSP, therefore serves as an intermediate for connecting the SSPto the target nucleic acid and the CSP hybridized to the target nucleicacid. The SSP may be prepared as described above. In another embodiment(FIG. 6C), the SSP used in the detection method comprises multiple setsof repeat sequences as well as a single stranded RNA sequence capable ofhybridizing to the bridge probe. A DNA oligonucleotide probe containingsequences complementary to the repeat sequences may be used to hybridizeto the SSP to generate the RNA-DNA duplex needed for signalamplification. In yet another embodiment (FIG. 6D), the bridge probecontains a poly(A) tail in addition to sequences which are capable ofhybridizing to the target nucleic acid. The SSP used in this examplecomprises poly(dT) DNA sequences. The bridge probe therefore is capableof hybridizing to the SSP via its poly(A) tail. A RNA probe comprisingpoly(A) sequences may be used to hybridize to the remaining poly(dT) DNAsequences within SSP to form a RNA-DNA duplex. The SSP comprisingpoly(dT) sequences and the RNA probe comprising poly(A) sequences arepreferably 100 to 5,000 bases long.

The SSP used in the detection method of the invention can be unmodified,or modified as with the CSP using methods described above and/or knownin the art. In a preferred embodiment, the SSP is a covalentlyunmodified probe.

It is understood that multiple CSPs and/or SSPs can be employed in thedetection method of the invention.

In another embodiment, an oligonucleotide probe comprising complementarysequences of two or more distinct regions of the target nucleic acid arefused together and used as the capture sequence probe in the method ofthe invention. Alternatively a single probe can be designed and producedwhich contains sequences complementary to single or multiple targetnucleic acids. This type of probe is also referred to herein as a“fused” CSP. As shown in Example 5, the fused capture sequence probeworks as effectively as the combination of two unfused CSPs when used atthe same concentration.

The nucleic acid probes of the invention may be produced by any suitablemethod known in the art, including for example, by chemical synthesis,isolation from a naturally-occurring source, recombinant production andasymmetric PCR (McCabe, 1990 In: PCR Protocols: A guide to methods andapplications. San Diego, Calif., Academic Press, 76-83). It may bepreferred to chemically synthesize the probes in one or more segmentsand subsequently link the segments. Several chemical synthesis methodsare described by Narang et al. (1979 Meth. Enzymol. 68:90), Brown et al.(1979 Meth. Enzymol. 68:109) and Caruthers et al. (1985 Meth. Enzymol.154:287), which are incorporated herein by reference. Alternatively,cloning methods may provide a convenient nucleic acid fragment which canbe isolated for use as a promoter primer. A double-stranded DNA probe isfirst rendered single-stranded using, for example, conventionaldenaturation methods prior to hybridization to the target nucleic acids.

Hybridization is conducted under standard hybridization conditionswell-known to those skilled in the art. Reaction conditions forhybridization of a probe to a nucleic acid sequence vary from probe toprobe, depending on factors such as probe length, the number of G and Cnucleotides in the sequence, and the composition of the buffer utilizedin the hybridization reaction. Moderately stringent hybridizationconditions are generally understood by those skilled in the art asconditions approximately 25° C. below the melting temperature of aperfectly base-paired double stranded DNA. Higher specificity isgenerally achieved by employing incubation conditions having highertemperatures, in other words more stringent conditions. Chapter 11 ofthe well-known laboratory manual of Sambrook et al., MOLECULAR CLONING:A LABORATORY MANUAL, second edition, Cold Spring Harbor LaboratoryPress, New York (1990) (which is incorporated by reference herein),describes hybridization conditions for oligonucleotide probes in greatdetail, including a description of the factors involved and the level ofstringency necessary to guarantee hybridization with specificity.Hybridization is typically performed in a buffered aqueous solution, forwhich conditions such as temperature, salt concentration, and pH areselected to provide sufficient stringency such that the probes hybridizespecifically to their respective target nucleic acid sequences but notany other sequence.

Generally, the efficiency of hybridization between probe and targetimprove under conditions where the amount of probe added is in molarexcess to the template, preferably a 2 to 10⁶ molar excess, morepreferably 10³ to 10⁶ molar excess. The concentration of each CSPprovided for efficient capture is at least 25 fmoles/ml (25 pM) in thefinal hybridization solution, preferably between 25 fmoles to 10⁴fmoles/ml (10 nM). The concentration of each SSP is at least 15 ng/ml inthe final hybridization solution, preferably 150 ng/ml. Table A showsthe conversion of SSP concentrations expressed in ng/ml to molar basis.

TABLE A Conversion of SSP Concentration From ng/ml to fmoles/ml SSPConcentration SSP Concentration in fmoles/ml (pM) in ng/ml SSP is a 3 kbRNA SSP is a 5 kb RNA  15 ng/ml 15.1 9 150 ng/ml 151 90 600 ng/ml 606364

Hybridization of the CSP and the SSP to the target nucleic acid may beperformed simultaneously or sequentially and in either order. In oneembodiment, hybridization of the CSP and hybridization of the SSP to thetarget nucleic acid are performed simultaneously. The hybrid formed isthen captured onto a solid phase coated with a substrate to which ligandattached to the CSP binds with specificity. In another embodiment,hybridization of the SSP to the target nucleic acid is performed afterthe hybridization of the CSP to the target nucleic acid. In this case,the CSP may be immobilized on a solid phase before or afterhybridization. In this embodiment, both the CSP and the target may bebound to the solid phase during the SSP hybridization reaction.

It will be understood by those skilled in the art that a solid phase ormatrix includes, for example, polystyrene, polyethylene, polypropylene,polycarbonate or any solid plastic material in the shape of plates,slides, dishes, beads, particles, cups, strands, chips and strips. Asolid phase also includes glass beads, glass test tubes and any otherappropriate glass product. A functionalized solid phase such as plasticor glass that has been modified so that the surface contains carboxyl,amino, hydrazide, aldehyde groups, nucleic acid or nucleotidederivatives can also be used. Any solid phase such as plastic or glassmicroparticles, beads, strips, test tubes, slides, strands, chips ormicrotiter plates can be used.

In one preferred embodiment, the CSP is labelled with biotin, andstreptavidin-coated or avidin-coated solid phase is employed to capturethe hybrid. More preferably, streptavidin-coated microtiter plates areused. These plates may be coated passively or covalently.

The captured hybrid may be detected by conventional means well-known inthe art, such as with a labelled polyclonal or monoclonal antibodyspecific for the hybrid, an antibody specific for one or more ligandsattached to the SSP, a labelled antibody, or a detectable modificationon the SSP itself.

One preferred method detects the captured hybrid by using ananti-RNA-DNA antibody. In this embodiment, the anti-RNA-DNA antibody ispreferably labelled with an enzyme, a fluorescent molecule or abiotin-avidin conjugate and is non-radioactive. The label can bedetected directly or indirectly by conventional means known in the artsuch as a colorimeter, a luminometer, or a fluorescence detector. Onepreferred label is, for example, alkaline phosphatase. Other labelsknown to one skilled in the art can also be employed as a means ofdetecting the bound double-stranded hybrid.

Detection of captured hybrid is preferably achieved by binding theconjugated antibody to the hybrid during an incubation step. Surfacesare then washed to remove any excess conjugate. These techniques areknown in the art. For example, manual washes may be performed usingeither an Eppendorf™ Repeat Pipettor with a 50 ml Combitip™ (Eppendorf,Hamburg, Germany), a Corning repeat syringe (Corning, Corning, N.Y.), asimple pump regulated by a variostat, or by gravity flow from areservoir with attached tubing. Commercially available tube washingsystems available from Source Scientific Systems (Garden Grove, Calif.)can also be used.

Bound conjugate is subsequently detected by a method conventionally usedin the art, for example, colorimetry or chemiluminescence as describedat Coutlee, et al. J. Clin. Microbiol. 27:1002-1007 (1989). Preferably,bound alkaline phosphatase conjugate is detected by chemiluminescence byadding a substrate which can be activated by alkaline phosphatase.Chemiluminescent substrates that are activated by alkaline phosphataseare well known in the art.

In another embodiment, the target specific hybrid capture method of theinvention employs blocker probes in addition to the CSP and SSP. Ablocker probe comprises sequences that are complementary to thesequences of the CSP. The sequence of a blocker probe is preferably atleast 75% complementary to the sequence of the CSP, more preferably,100% complementary to the CSP. The addition of the blocker probes to thehybridization reaction mixture prevents non-hybridized CSP fromhybridizing to cross-reactive nucleic acid sequences present in thetarget and therefore increases the specificity of the detection.

The blocker probe is generally at least 5 bases long, preferably 12bases long. The concentration of the blocker probe in the hybridizationreaction is preferably in excess to that of the CSP and SSP. Preferably,the blocker probe is present in a 2-fold molar excess, although, it maybe present in an up to 10.000-fold molar excess. The blocker probes canbe DNA, RNA, peptide nucleic acids (PNAs) or other nucleic acidanalogues.

In one embodiment, blocker probes complementary to the full-length ornear full-length of the CSP are used. Following the reaction in whichthe hybrid between CSP, SSP and the target nucleic acid is formed, oneor more blocker probes may be added to the reaction and thehybridization is continued for a desired time. The hybridizationproducts are then detected as described above.

In another embodiment, blocker probes complementary to only a portion ofthe CSP and are shorter than the CSP are used. These blocker probes havea lower melting temperature than that of the CSP. Preferably, themelting temperature of the blocker probe is 10 degrees lower than thatof the CSP. In this case, the blocker probe is preferably added to thetarget nucleic acids simultaneously with the CSP and the SSP. Since theblocker probe has a lower melting temperature than the CSP, the initialtemperature for hybridization is chosen such that the blocker probe doesnot interfere with the hybridization of the CSP to its target sequences.However, when the temperature of the hybridization mixtures is adjustedbelow the temperature used for target hybridization, the blocker probehybridizes to the CSP and effectively blocks the CSP from hybridizing tocross-reactive nucleic acid sequences. For example, when thehybridization products are incubated at room temperature on astreptavidin-coated microtiter plate during hybrid capture, the blockerprobes may be added.

The following examples illustrate use of the present amplificationmethod and detection assay and kit. These examples are offered by way ofillustration, and are not intended to limit the scope of the inventionin any manner. All references described herein are expresslyincorporated in toto by reference.

EXAMPLE 1 Target-Specific Hybrid Capture (TSHC) Assay Protocol

Herpes Simplex Virus 1 (HSV-1) and Herpes Simplex Virus 2 (HSV-2) viralparticles of known concentration (Advanced Biotechnologies, Inc.,Columbia, Md.) or clinical samples were diluted using either NegativeControl Media (Digene Corp., Gaithersburg, Md.) or Negative CervicalSpecimens (Digene). Various dilutions were made and aliquoted intoindividual microfuge tubes. A half volume of the Denaturation Reagent5100-0431 (Digene) was added. Test samples were incubated at 65° C. for45 minutes for denaturation of nucleic acids in the samples.

Following denaturation, a hybridization solution containing signalsequence probes (SSPs) (600 ng/ml each) and capture sequence probes(CSPs) (2.5 pmoles/ml each) was added to the sample, and incubated at74° C. for 1 hour. Blocker probes in a solution containing one volume of4× Probe Diluent (Digene), one volume of Denaturation Reagent and twovolumes of the Negative Control Media were then added to thehybridization mixture and incubated at 74° C. for 15 minutes.

In a second series of experiments, following denaturation of nucleicacids, a hybridization mixture containing SSPs (600 ng/ml each), CSPs(2.5 pmoles/ml each), and blocker probes (250 pmoles/ml each) was addedto the samples and incubated for one hour at 74° C.

Tubes containing reaction mixtures were cooled at room temperature for 5minutes, and aliquots were taken from each tube and transferred toindividual wells of a 96-well streptavidin capture plate (Digene). Theplates were shaken at 1100 rpms for 1 hour at room temperature. Thesupernatants were then decanted and the plates were washed twice withSNM wash buffer (Digene) and inverted briefly to remove residual washbuffer. The alkaline-phosphatase anti-RNA/DNA antibody DR-1 (Digene) wasthen added to each well and incubated 30 minutes at room temperature.The wells were then subjected to multiple wash steps which include: 1)three washes with Sharp wash buffer (Digene) at room temperature; 2)incubation of the plate with the Sharp wash buffer for 10 minutes at 60°C. on a heat block; 3) two washes with the Sharp wash buffer at roomtemperature; and 4) one wash with the SNM wash buffer (Digene) at roomtemperature. Following removal of the residual liquid, luminescentsubstrate 5100-0350 (Digene) was added to each well and incubated for 15minutes at room temperature. The individual wells were then read on aplate luminometer to obtain the relative light unit (RLU) signal.

Solutions containing Negative Control Media or known HSV NegativeCervical Specimens were used as negative controls for the test samples.The signal to noise ratio (S/N) was calculated as the ratio of theaverage RLU obtained from a test sample to the average RLU of thenegative control. The signal to noise ratio was used as the basis fordetermining capture efficiency and the detection of target nucleicacids. A S/N value of 2 or greater was arbitrarily assigned as apositive signal while a S/N values less than 2 was considered negative.The coefficient of variation (CV) which is a determination of thevariability of the experiment within one sample set was calculated bytaking the standard deviation of the replicates, dividing them by theaverage and multiplying that value by 100 to give a percent value.

The capture sequence probes and the blocker probes used in experimentsdescribed in Examples 2-13 were synthesized using the method describedby Cook et al. (1988 Nucl. Acid. Res., 16: 4077-95). Unless otherwisenoted, the capture sequence probes used in the experiments describedherein were labeled with biotins at their 5′ and 3′ ends.

The signal sequence probes used in experiments described in Examples2-13 are RNA probes. These probes were prepared using the methoddescribed by Yisraeli et al. (1989, Methods in Enzymol., 180: 42-50).

EXAMPLE 2

The following tables describe the various probes used in experimentsdescribed in Examples 3-13.

TABLE 1 HSV-1 Clones from which HSV-1 Probes are derived SequenceLocation Clone Name Host Vector Cloning Site(s) Insert Size (bp) withinHSV-1 RH3 Dgx3 Hind III, Eco RI 5720 39850-45570 R10 Blue Script SK+ EcoRI 4072 64134-68206 RH5B Blue Script SK+ Eco RV, Eco RI 4987105108-110095 H19 Blue Script SK+ Hind III 4890 133467-138349

TABLE 2 Clones from which HSV-2 Probes are derived Sequence LocationClone Name Host Vector Cloning Site(s) Insert Size (bp) in HSV-2 E4ABlue Script SK+ Bam HI 3683 23230-26914 E4B Blue Script SK+ Bam HI EcoRI 5600 26914-32267 I8 Blue Script SK+ Hind III 2844 41624-44474 EI8Dgx3 Hind III, Eco RI 3715 44474-48189 4L Blue Script KS+ Bam HI, Eco RI4313 86199-90512

TABLE 3 Capture Sequence Probes for HSV-1 Location Size within ProbeSequence (bp) HSV-1 TS-1 (TTATTATTA)CGTTCATGTCGGCAAACAGCT 24105040-105063 CGT(TTATTATTA) [SEQ ID NO:1] TS-2(TTATTATTA)CGTCCTGGATGGCGATACGGC 21 110316-110336 (TTATTATTA) [SEQ IDNO:2] VH-3 CGTCCTGGATGGCGATACGGC 21 110316-110336 [SEQ ID NO:3] NC-1CGTTCATGTCGGCAAACAGCTCGT 24 105040-105063 [SEQ ID NO:4] VH-4CGTTCATGTCGGCAAACAGCTCGT- 45 105040-105063; (fusionCGTCCTGGATGGCGATACGGC 110316-110336 of VH3, [SEQ ID NO:5] NC-1) HZ-1GATGGGGTTATTTTTCCTAAGATGGGGC 34 133061-133094 GGGTCC [SEQ ID NO:6] VH-2TACCCCGATCATCAGTTATCCTTAAGGT 28 138367-138394 [SEQ ID NO:7] FD-1AAACCGTTCCATGACCGGA 19  39281-39299 [SEQ ID NO:8] RA-2ATCGCGTGTTCCAGAGACAGGC 22  39156-39177 [SEQ ID NO:9] NC-2CAACGCCCAAAATAATA 17  46337-46353 [SEQ ID NO:10] FD-2GTCCCCGAaCCGATCTAGCG (note small 20  45483-45502 cap a is mutated base)[SEQ ID NO:11] RA-4 CGAACCATAAACCATTCCCCAT 22  46361-46382 [SEQ IDNO:12] ON-3 CACGCCCGTGGTTCTGGAATTCGAC 25  64105-64129 [SEQ ID NO:13]HZ-2 (TTTATTA)GATGGGGTTATTTTTCCTAAGAT 34 133061-133094 GGGGCGGGTCC [SEQID NO:14] ZD-1 GGTTATTTTTCCTAAG 16 133064-133079 [SEQ ID NO:15] ZD-2(ATTATT)GGTTATTTTTCCTAAG(ATTATT) 16 133064-133079 [SEQ ID NO:16] F6RACGACGCCCTTGACTCCGATTCGTCATCGGAT 40  87111-87150 GACTCCCT [SEQ ID NO:17]BRH19 ATGCGCCAGTGTATCAATCAGCTGTTTCGGGT 32 133223-133254 [SEQ ID NO:18]F15R CAAAACGTCCTGGAGACGGGTGAGTGTCGGC 38 141311-141348 GAGGACG [SEQ IDNO:19] VH-1 GTCCCCGACCCGATCTAGCG 20  45483-45502 [SEQ ID NO:20] ON-4GCAGACTGCGCCAGGAACGAGTA 23  68404-68426 [SEQ ID NO:21] PZ-1GTGCCCACGCCCGTGGTTCTGGAATTCGACAG 35  64105-64139 CGA [SEQ ID NO:22] PZ-2GCAGACTGCGCCAGGAACGAGTAGTTGGAGT 35  68404-68438 ACTG [SEQ ID NO:23] FG-2AAGAGGTCCATTGGGTGGGGTTGATACGGGA 36 105069-105104 AAGAC [SEQ ID NO:24]FG-3 CGTAATGCGGCGGTGCAGACTCCCCTG 27 110620-110646 [SEQ ID NO:25] FG-4CCAACTACCCCGATCATCAGTTATCCTT 39 138362-138400 AAGGTCTCTTG [SEQ ID NO:26]Hsv1-LF15R (AAAAAAAAA)CAAAACGTCCTGGAGACGGGT 38 141311-141348 (SH-3)GAGTGTCGGCGAGGACG [SEQ ID NO:27] Hsv1-F15-2BCAAAACGTCCTGGAGACGGGTGAGTGTCGGC 38 141311-141348 (GZ-1) GAGGACG [SEQ IDNO:28] Hsv1-F15-3B CAAAACGTCC-bio-U-GGAGACGGGTGAG 38 141311-141348(GZ-2) TG-bio-U-CGGCGAGGACG [SEQ ID NO:29] * Sequences in parenthesesare “tail” sequences not directed at HSV.

TABLE 4 Blocker Probes for HSV-1 Capture Probe to Size which it ProbeSequence (bp) hybridizes EA-1 AGGAAAAATAACCCCATC 18 HZ-1 [SEQ ID NO:30]EA-2 GACCCGCCCCATCTT 15 HZ-1 [SEQ ID NO:31] ZD-3GGACCCGCCCCATCTTAGGAAAAATAA 34 HZ-1 CCCCATC [SEQ ID NO:32] NG-7AAAAATAACCCCA 13 HZ-1 [SEQ ID NO:33] NG-8 CGCCCCATCTT 11 HZ-1 [SEQ IDNO:34] NG-4 CCATCTTAGGAAAAA 15 HZ-1 [SEQ ID NO:35] GP-1 ATAACTGATGATCGG15 VH-Z [SEQ ID NO:36] EA-3 CCACCCAATGGACCTC 16 FG-2 [SEQ ID NO:37] EA-4GTCTTTCCCGTATCAACC 18 FG-2 [SEQ ID NO:38] EB-7 CGCCGCATTACG 12 FG-3 [SEQID NO:39] EB-8 AGGGGAGTCTGC 12 FG-3 [SEQ ID NO:40] GP-3 CTGTTTGCCGACA 13VH-4 [SEQ ID NO:41] GP-4 TATCGCCATCCAG 13 VH-4 [SEQ ID NO:42] EB-9ATGATCGGGGTAGT 14 FG-4 [SEQ ID NO:43] EB-10 AGAGACCTTAAGGATA 16 FG-4[SEQ ID NO:44] NG-1 ATTCCAGAACCACGG 15 ON-3 [SEQ ID NO:45] NG-2TTCCAGAACCACG 13 ON-3 [SEQ ID NO:46] NG-3 TCCAGAACCAC 11 ON-4 [SEQ IDNO:47] GP-5 GTTCCTGGCGCAG 13 ON-4 [SEQ ID NO:48] GP-6 TTCCTGGCGCAG 12ON-4 [SEQ ID NO:49]

TABLE 5 Capture Sequence Probes for HSV-2 Location Size within ProbeSequence (bp) HSV-2 NF-1 GCCCGCGCCGCCAGCACTACTTTC 24 41610-41587 [SEQ IDNO:50] FG-1 AAACGTTGGGAGGTGTGTGCGTCATCC 35 48200-48234 TGGAGCTA [SEQ IDNO:51] LE-3 GACCAAAACCGAGTGAGGTTCTGTGT 26 48732-48757 [SEQ ID NO:52]NF-2 AAACGTTGGGAGGTGTGTGCGTCA 24 48200-48223 [SEQ ID NO:53] RA-3TGCTCGTCACGAAGTCACTCATG 23 22756-22734 [SEQ ID NO:54] ON-2CATTACTGCCCGCACCGGACC 21 23862-23842 [SEQ ID NO:55] LE-1GCCGTGGTGTTCCTGAACACCAGG 24 27666-27643 [SEQ ID NO:56] LE-4AGTCAGGGTTGCCCGACTTCGTCAC 25 22891-22867 [SEQ ID NO:57] NF-3CAGGCGTCCTCGGTCTCGGGCGGGGC 26 32847-32822 [SEQ ID NO:58] NF-4CCCACGTCACCGGGGGCCCC 20 26743-26724 [SEQ ID NO:59] LE-2GCCGGTCGCGTGCGACGCCCAAGGC 25 33130-33106 [SEQ ID NO:60] SG-3CCGACGCGTGGGTATCTAGGGGGTCG 26 90559-90534 [SEQ ID NO:61] SG-4CGGGACGGCGAGCGGAAAGTCAACGT 26 86194-86169 [SEQ ID NO:62]

TABLE 6 Blocker Probes for HSV-2 Capture Probe to which it Probe Sizehybri- Name Sequence (bp) dizes HX-4 GGCGCGGGC [SEQ ID NO:63] 9 NF-1HX-5 GAAAGTAGTGCTGGC [SEQ ID NO:64] 15 NF-1 GP-7 TGCTGGCGGCG [SEQ IDNO:65] 11 NF-1 AZ-3 ACACCTCCCAACG [SEQ ID NO:66] 13 FG-1 AZ-4CTCCAGGATGACG [SEQ ID NO:67] 13 FG-1 GR-1 TCGGTTTTGGTC [SEQ ID NO:68] 12LE-3 GR-2 ACACAGAACCTCA [SEQ ID NO:69] 13 LE-3 GP-8 CACACACCTCCCA [SEQID NO:70] 13 NF-2 BR-10 CGACCCCCTAGATA [SEQ ID NO:71] 14 SG-3 BR-11CCACGCGTCGG [SEQ ID NO:72] 11 SG-3 HX-6 ACGTTGACTTTCCGC [SEQ ID NO:73]15 SG-4 BR-15 CGCCGTCCCG [SEQ ID NO:74] 10 SG-4

TABLE 7 Capture Sequence Probes for HPV HPV Type and Size Sequence ProbeSequence (bp) Location ZL-1 GTACAGATGGTACCGGGGTTGTAGAAGTATCTG 33 HPV16[SEQ ID NO:75] 5360-5392 ZL-4 CTGCAACAAGACATACATCGACCGGTCCACC 31 HPV16[SEQ ID NO:76] 495-525 DP-1 GAAGTAGGTGAGGCTGCATGTGAAGTGGTAG 31 HPV16[SEQ ID NO:77] 5285-5315 DP-4 CAGCTCTGTGCATAACTGTGGTAACTTTCTGGG 33 HPV16[SEQ ID NO:78] 128-160 SH-1 GAGGTCTTCTCCAACATGCTATGCAACGTCCTG 33 HPV31[SEQ ID NO:79] 505-537 SH-4 GTGTAGGTGCATGCTCTATAGGTACATCAGGCC 33 HPV31[SEQ ID NO:80] 5387-5419 VS-1 CAATGCCGAGCTTAGTTCATGCAATTTCCGAGG 33 HPV31[SEQ ID NO:81] 132-164 VS-4 GAAGTAGTAGTTGCAGACGCCCCTAAAGGTTGC 33 HPV31[SEQ ID NO:82] 5175-5207 AH-1 GAACGCGATGGTACAGGCACTGCAGGGTCC 30 HPV18[SEQ ID NO:83] 5308-5337 AH-2 GAACGCGATGGTACAGGCACTGCA 24 HPV18 [SEQ IDNO:84] 5314-5337 AL-1 ACGCCCACCCAATGGAATGTACCC 24 HPV18 [SEQ ID NO:85]4451-4474 PA-4 TCTGCGTCGTTGGAGTCGTTCCTGTCGTGCTC 32 HPV18 [SEQ ID NO:86]535-566 18-1AB (TTATTATTA)CTACATACATTGCCGCCATGTTCG 36 HPV18 CCA1369-1395 [SEQ ID NO:87] 18-2AB (TTATTATTA)TGTTGCCCTCTGTGCCCCCGTTGT 46HPV18 CTATAGCCTCCGT 1406-1442 [SEQ ID NO:88] 18-3AB(TTATTATTA)GGAGCAGTGCCCAAAAGATTAAA 38 HPV18 GTTTGC 7524-7552 [SEQ IDNO:89] 18-4AB (TTATTATTA)CACGGTGCTGGAATACGGTGAGG 37 HPV18 GGGTG3485-3512 [SEQ ID NO:90] 18-5AB (TTATTATTA)ACGCCCACCCAATGGAATGTACCC 33HPV18 [SEQ ID NO:91] 4451-4474 18-6AB(TTATTATTA)ATAGTATTGTGGTGTGTTTCTCAC 35 HPV18 AT  81-106 [SEQ ID NO:92]18-7AB (TTATTATTA)GTTGGAGTCGTTCCTGTCGTG 30 HPV18 [SEQ ID NO:93] 538-55818-8AB (TTATTATTA)CGGAATTTCATTTTGGGGCTCT 31 HPV18 [SEQ ID NO:94] 634-655PE-1 GCTCGAAGGTCGTCTGCTGAGCTTTCTACTACT 33 HPV18 [SEQ ID NO:95] 811-843PZ-2 GCGCCATCCTGTAATGCACTTTTCCACAAAGC 32 HPV45 [SEQ ID NO:96]  77-108PZ-5 TAGTGCTAGGTGTAGTGGACGCAGGAGGTGG 31 HPV45 [SEQ ID NO:97] 5295-5325CS-1 GGTCACAACATGTATTACACTGCCCTCGGTAC 32 HPV45 [SEQ ID NO:98] 500-531CS-4 CCTACGTCTGCGAAGTCTTTCTTGCCGTGCC 31 HPV45 [SEQ ID NO:99] 533-563PF-1 CTGCATTGTCACTACTATCCCCACCACTACTTTG 34 HPV45 [SEQ ID NO:100]1406-1439 PF-4 CCACAAGGCACATTCATACATACACGCACGCA 32 HPV45 [SEQ ID NO:101]7243-7274 PA-1 GTTCTAAGGTCCTCTGCCGAGCTCTCTACTGTA 33 HPV45 [SEQ IDNO:102] 811-843 45-5AB (TTATTATTA)TGCGGTTTTGGGGGTCGACGTGGA 36 HPV45 GGC3444-3470 [SEQ ID NO:103] 45-6AB (TTATTATTA)AGACCTGCCCCCTAAGGGTACATA 36HPV45 GCC 4443-4469 [SEQ ID NO:104] 45-8AB(TTATTATTA)CAGCATTGCAGCCTTTTTGTTACT 49 HPV45 TGCTTGTAATAGCTCC 1477-1516[SEQ ID NO:105] 45-9AB (TTATTATTA)ATCCTGTAATGCACTTTTCCACAAA 34 HPV45[SEQ ID NO:106]   79-103 45-10AB (TTATTATTA)GCCTGGTCACAACATGTATTAC 31HPV45 [SEQ ID NO:107] 514-535 45-11AB(TTATTATTA)CAGGATCTAATTCATTCTGAGGTT 33 HPV45 [SEQ ID NO:108] 633-656ON-1 TGCGGTTTTGGGGGTCGACGTGGAGGC 27 HPV45 [SEQ ID NO:109] 3444-3470 *Sequences in parentheses are “tail” sequences not directed at HSV.

TABLE 8 Blocker Probes For HPV Capture Probe to Size which it ProbeSequence (bp) hybridizes PV-FD-1 GCCTCCACGTCGAC 14 ON-1/45-5AB [SEQ IDNO:110] PV-FD-2 CCCCAAAACCG 11 ON-1/45-5AB [SEQ ID NO:111] PV-FD-3GGTACATTCCATTGGG 16 18-5AB/AL-1 [SEQ ID NO:112] PV-FD-4 TGGGCGTTAATAATAA16 18-5AB [SEQ ID NO:113] AH-3 ACCATCGCGTTC 12 AH-2 [SEQ ID NO:114] AH-4GGACCCTGCAGTGC 14 AH-1 [SEQ ID NO:115] AH-5 CTGTACCATCGCGTT 3′ 15 AH-1[SEQ ID NO:116] AH-6 TGCAGTGCCTGT 12 AH-2 [SEQ ID NO:117] PZ-1CCACCTCCTGCGT 13 PZ-5 [SEQ ID NO:118] PZ-3 ATTACAGGATGGCGC 15 PZ-2 [SEQID NO:119] PZ-4 GCTTTGTGGAAAAGTG 16 PZ-2 [SEQ ID NO:120] PZ-6CCACTACACCTAGCACTA 18 PZ-5 [SEQ ID NO:121] ZL-2 CAGATACTTCTACAACC 17ZL-1 [SEQ ID NO:122] ZL-3 CCGGTACCATCTGTAC 16 ZL-1 [SEQ ID NO:123] ZL-5GGTGGACCGGTCG 13 ZL-4 [SEQ ID NO:124] ZL-6 ATGTATGTCTTGTTGCAG 18 ZL-4[SEQ ID NO:125] DP-2 CTACCACTTCACATGC 16 DP-1 [SEQ ID NO:126] DP-3AGCCTCACCTACTTC 15 DP-1 [SEQ ID NO:127] DP-5 CCCAGAAAGTTACCAC 16 DP-4[SEQ ID NO:128] DP-6 AGTTATGCACAGAGCT 16 DP-4 [SEQ ID NO:129] SH-2CAGGACGTTGCATAGC 16 SH-1 [SEQ ID NO:130] SH-3 ATGTTGGAGAAGACCTC 17 SH-1[SEQ ID NO:131] SH-5 GGCCTGATGTACCTATA 17 SH-4 [SEQ ID NO:132] SH-6GAGCATGCACCTACAC 16 SH-4 [SEQ ID NO:133] VS-2 CTCGGAAATTGCATG 15 VS-1[SEQ ID NO:134] VS-3 AACTAAGCTCGGCATT 16 VS-1 [SEQ ID NO:135] VS-5GCAACCTTTAGGGG 14 VS-4 [SEQ ID NO:136] VS-6 CGTCTGCAACTACTACTTC 19 VS-4[SEQ ID NO:137] CS-2 GTACCGAGGGCAGT 14 CS-1 [SEQ ID NO:138] CS-3GTAATACATGTTGTGACC 18 CS-1 [SEQ ID NO:139] CS-5 GGCACGGCAAGAAA 14 CS-4[SEQ ID NO:140] CS-6 GACTTCGCAGACGTAGG 17 CS-4 [SEQ ID NO:141] PF-2CAAAGTAGTGGTGGG 15 PF-1 [SEQ ID NO:142] PF-3 GATAGTAGTGACAATGCAG 19 PF-1[SEQ ID NO:143] PF-5 TGCGTGCGTGTATGTA 16 PF-4 [SEQ ID NO:144] PF-6TGAATGTGCCTTGTGG 16 PF-4 [SEQ ID NO:145] PE-2 AGTAGTAGAAAGCTCAGC 18 PE-1[SEQ ID NO:146] PE-3 AGACGACCTTCGAGC 15 PE-1 [SEQ ID NO:147] PA-2TACAGTAGAGAGCTCGG 17 PA-1 [SEQ ID NO:148] PA-3 CAGAGGACCTTAGAAC 16 PA-1[SEQ ID NO:149] PA-5 GAGCACGACAGGAACG 16 PA-4 [SEQ ID NO:150] PA-6ACTCCAACGACGCAGA 16 PA-4 [SEQ ID NO:151]

EXAMPLE 3 Effect of the Extent of Biotin Labeling on Capture Efficiency

Tests were conducted to determine the optimal number of biotin labelsper capture sequence probe for TSHC detection. The general TSHC methoddescribed in Example 1 was employed. The capture efficiency of capturesequence probe F15R labelled with one, two, or three biotins, measuredby signal to noise ratio (S/N), were tested. The signal sequence probeemployed was H19. As shown in Table 9, two biotins per capture sequenceprobe were sufficient for optimal capture efficiency. Greater than a 50%increase in S/N was observed using capture sequence probe with twobiotin labels compared to the single biotin labeled capture sequenceprobe. The addition of a third biotin label to the capture sequenceprobe resulted in a decrease in S/N relative to the two-biotin labeledcapture sequence probe.

TABLE 9 Effect of the Extent of Biotin Labeling on Capture Efficiency #Biotins HSV-1/well RLU CV S/N One 0 54 3% 1.0 One 4.5 × 10{circumflexover ( )}3 236 2% 4.4 One 4.5 × 10{circumflex over ( )}4 1861 3% 34.5One 4.5 × 10{circumflex over ( )}5 15633 7% 289.5 Two 0 46 3% 1.0 Two4.5 × 10{circumflex over ( )}3 296 10%  6.4 Two 4.5 × 10{circumflex over( )}4 2558 1% 55.6 Two 4.5 × 10{circumflex over ( )}5 23369 4% 508.0Three 0 44 22%  1.0 Three 4.5 × 10{circumflex over ( )}3 243 6% 5.5Three 4.5 × 10{circumflex over ( )}4 1820 2% 51.4 Three 4.5 ×10{circumflex over ( )}5 18581 8% 422.3

EXAMPLE 4 Effect of the Distance between the CSP and the SSP TargetSites on Capture Efficiency

The effect of the distance between capture sequence probe (CSP) andsignal sequence probe (SSP) hybridization sites on a HSV-1 targetnucleic acid on capture efficiency was evaluated. CSPs that hybridize toHSV-1 nucleic acid sequences which are located 0.2 kb, 3 kb, 18 kb, 36kb and 46 kb from the site of SSP hybridization were tested. The generalTSHC method described in Example 1 was employed. The captureefficiencies were 100%, 50%, 30%, 19% and 7%, respectively (Table 10). Asteady decline in relative capture efficiencies was observed as thedistance increased from 0.2 Kb to 46 Kb.

TABLE 10 Effect of Distance between Target Sites on Capture EfficiencyDistance Between Relative Capture CSP SSP Target Site Efficiency BRH19H19 0.2 Kb 100%  F15R H19 3 Kb 50% F6R RH5B 18 Kb 30% F15R RH5B 36 Kb19% F6R H19 46 Kb  7%

EXAMPLE 5 Effect of Fused Capture Sequence Probe on TSHC Detection ofHSV-1

The binding capacity of streptavidin plates was determined to beapproximately 2 pmoles of doubly-biotinylated CSPs per well. Since theCSPs are doubly biotin-labeled, a maximum of 8 CSPs (2 CSPs per SSP) ispreferred in order not to exceed the binding capacity of the wells. Anyincrease in biotin-labeled capture sequence probe above the statedcapacity resulted in a decrease in signal, the so-called “hook effect.”In order to avoid this “hook effect” and still permit the use of greaterthan four SSP-CSP combinations, the effect of synthesizingoligonucleotides that contained the sequences of two CSPs fused together(5′ and 3′ sites) was tested. The fused capture sequence probes mayfunction independently to drive hybridization to the unique targetsites. In another embodiment, the fused probes may bind to two targetsites with the second hybridization favored, since it is essentially auni-molecular reaction with zero order kinetics once the probe hashybridized to the first site. The hybridization may be determined by oneor both mechanisms. Previous experiments showed that two CSPs, VH3 andNC-1, when used together, gave approximately twice the S/N as theindividual CSPs. Unfused capture sequence probes VH-3 and NC-1 were usedat 2.5 pmoles/ml each for a total concentration of 5 pmoles/ml, fusedprobe VH-4 (fusion of VH-3 and NC-1) was used at 2.5 pmole/ml. As shownin Table 11, the fused probe was as effective as the combination of thetwo unfused probes. Therefore, TSHC detection using fused capturesequence probes permits the number of nucleic acid sequences targeted bythe signal sequence probe to be at least doubled without exceeding theplate biotin-binding capacity. The experiment also demonstrates the lackof cross-reactivity of HSV-2 at 107 genomes as shown by the S/N lessthan 2.0.

TABLE 11 Comparison of Fused v. Unfused Capture Sequence Probes in TSHCDetection of HSV-1 SSP CSP Viral Particles/ml RLU CV S/N RH5B VH-3, NC-10 94 14%  1.0 RH5B VH-3, NC-1 10{circumflex over ( )}4 HSV-1 164 5% 1.7RH5B VH-3, NC-1 10{circumflex over ( )}5 HSV-1 1003 4% 10.7 RH5B VH-3,NC-1 10{circumflex over ( )}7 HSV-2 125 6% 1.3 RH5B VH-4 (fused) 0 9710%  1.0 RH5B VH-4 (fused) 10{circumflex over ( )}4 HSV-1 181 3% 1.9RH5B VH-4 (fused) 10{circumflex over ( )}5 HSV-1 1070 2% 11.0 RH5B VH-4(fused) 10{circumflex over ( )}7 HSV-2 140 5% 1.4

EXAMPLE 6 Capture Efficiency of Various CSPs and SSPs in TSHC Detectionof HSV-1

The capture efficiency of capture sequence probes (CSPs) for each of thefour HSV-1 specific signal sequence probes (SSPs), H19, RH5B, RH3 andRIO, in the detection of HSV-1 by TSHC were evaluated. The criteria usedfor designing the capture sequence probes were: 1) the CSP hybridizationsite is within 1 kb either 5′ or 3′ of the SSP hybridization site on theHSV-1 nucleic acid sequence, preferably within 0.5 kb; and 2) the CSPscontain sequences that are unique to HSV-1, with no stretches ofsequence homology to HSV-2 greater than 10 bases. The CSPs were designedto target the 5′ and 3′ regions adjacent to the SSP hybridization site,preferably with a 5′ CSP and a 3′ CSP for each SSP. The Omiga software(Oxford Molecular Group, Campbell, Calif.) was instrumental in theidentification of such sites. The melting temperature (Tm) of the CSPswas designed to be between 70° C. to 85° C., to conform to the 70° C. to75° C. hybridization temperature used in Hybrid Capture II (HCII) assayfor HSV (Digene). The general TSHC method described in Example 1 wasemployed. Eleven CSPs (which bind to 6 different sites) for H19, sixCSPs (which bind to three unique sites) for RH5B, six CSPs (which bindto six unique sites) for RH3, and two CSPs for R10 were tested. As shownin Table 12, efficient capture sequence probes were found for signalsequence probes H19, RH5B and R10.

TABLE 12 CSPs and SSPs for TSHC Detection of HSV-1 SSP CSP Cap % R10ON-3 100%  R10 ON-3 80% RH5B TS-1 50% RH5B NC-1 75% RH5B VH-4 130%  RH5BTS-2 25% RH5B VH-3 50% H19 HZ-1 50% H19 HZ-2 20% H19 ZD-1 40% H19 ZD-220% H19 BRH19 70% H19 VH-2 70% H19 F15R 25%

EXAMPLE 7 Capture Efficiency of Various CSPs and SSPs in TSHC Detectionof HSV-2

The capture efficiency of capture sequence probes (CSPs) for each of thefour HSV-2 specific signal sequence probes (SSPs), E4A, E4B, Ei8, andi8, in the detection of HSV-2 by TSHC were evaluated. HSV-2 specificcapture sequence probes (CSPs) were designed based on the same criteriaas the HSV-1 CSPs except for the requirement that they be HSV-2specific. Four CSPs for E4A, three CSPs for E4B, and two CSPs each forEi8 and i8 were tested. The general TSHC method described in Example 1was employed. As shown in Table 13, efficient capture sequence probeswere found for i8 and Ei8.

TABLE 13 CSPs and SSPs for TSHC Detection of HSV-2 SSP CSP Cap % I8 NF-1100%  Ei8 NF-2 50% Ei8 LE-3 45%

EXAMPLE 8 Effect of Blocker Probes on HSV-1 and HSV-2 Detection

In an attempt to reduce cross-reactivity of TSHC while allowing thecapture step to take place at room temperature, methods using blockerprobes were developed. Blocker probes comprise sequences that arecomplementary to the capture sequence probes (CSPs) used for detection.These experiments were designed to prevent non-specific hybridization ofthe CSPs to non-targeted nucleic acids present in the sample under thelower stringency conditions, a situation often encountered during theroom temperature capture step.

In one method, blocker probes that are complementary to the full lengthor nearly the full length of the capture sequences probe were used. Theblocker probes were added to the reaction mixture in 10-fold excessrelative to the CSP after hybridization of the CSP and the SSP to thetarget DNA molecule has occurred. Since the blocker probes have similarmelting temperature as the CSPs, the CSPs were hybridized to the targetnucleic acids first to prevent hybridization of the blocker probes tothe CSPs before the hybridization of the CSPs to the target nucleicacids occurred. As shown in Table 14, the addition of the blocker probesresulted in a dramatic reduction in cross-reactivity while these probeshad no effect on the sensitivity of HSV-1 detection. The S/N for thedetection of cross-reactive HSV-2 (107 viral particles/ml) decreasedfrom 5.0 to 0.8 when the blocker probes were used.

In another method, blocker probes that are complementary to only aportion of the CSPs and are shorter than the CSPs were used. The blockerprobes were designed to have melting temperatures above room temperaturebut at least 10° C. below the hybridization temperature of CSPs to thetarget nucleic acids. Since these blocker probes hybridize to the CSPsat temperature below the CSP hybridization temperature to the targetnucleic acids, the blocker probes may be added to the reaction at thesame time as the CSP and SSP without effecting the hybridizationefficiency of the CSPs to the target nucleic acid. These shorter blockerprobes function during the room temperature capture step by hybridizingto the CSPs at the lower temperatures that are encountered during theroom temperature capture step. As shown in Table 15, the addition ofeither single or paired shorter blocker probes in 100-fold excessrelative to the CSPs resulted in a dramatic reduction incross-reactivity but had no effect on sensitivity of HSV-1 detection.The S/N for detecting cross-reactive HSV-2 (107 viral particles/ml)without the blocker probes was 10.6, but was reduced to less than orequal to 1.5 with the addition of the blocker probes.

Therefore, both methods utilizing blocker probes provide a substantialreduction in cross-reactivity. The second method utilizing blockerprobes with lower melting temperature may be preferred because theaddition of blocker probes at the same time as the capture sequenceprobe eliminates the need for an extra step for the detection method.

TABLE 14 Effect of Blocker Probes Added Post Capture probe hybridizationon TSHC 100x Blocker SSP CSP Probe Viral Particles/ml RLU CV S/N H19HZ-1 None 0 66 7% 1.0 H19 HZ-1 None 10{circumflex over ( )}5 HSV-1 2465% 3.7 H19 HZ-1 None 10{circumflex over ( )}6 HSV-1 1998 2% 30.3 H19HZ-1 None 10{circumflex over ( )}7 HSV-2 327 2% 5.0 H19 HZ-1 ZD-3 0 603% 1.0 H19 HZ-1 ZD-3 10{circumflex over ( )}5 HSV-1 267 4% 4.5 H19 HZ-1ZD-3 10{circumflex over ( )}6 HSV-1 2316 6% 38.6 H19 HZ-1 ZD-310{circumflex over ( )}7 HSV-2 49 2% 0.8

TABLE 15 Effect of Blocker Probes Added Simultaneously with the CaptureProbes on TSHC Detection of HSV-1 10x Blocker SSP CSP Probe ViralParticle/ml RLU CV S/N H19 HZ-1 none 0 38 15%  1.0 H19 HZ-1 none10{circumflex over ( )}4 HSV-1 71 2% 1.9 H19 HZ-1 none 10{circumflexover ( )}5 HSV-1 389 12%  10.2 H19 HZ-1 none 10{circumflex over ( )}7HSV-2 401 18%  10.6 H19 HZ-1 NG-4 0 39 8% 1.0 H19 HZ-1 NG-410{circumflex over ( )}4 HSV-1 82 5% 2.1 H19 HZ-1 NG-4 10{circumflexover ( )}5 HSV-1 411 18%  10.5 H19 HZ-1 NG-4 10{circumflex over ( )}7HSV-2 57 15%  1.5 H19 HZ-1 EA-1, EA-2 0 37 0% 1.0 H19 HZ-1 EA-1, EA-210{circumflex over ( )}4 HSV-1 75 8% 2.0 H19 HZ-1 EA-1, EA-210{circumflex over ( )}5 HSV-1 419 8% 11.3 H19 HZ-1 EA-1, EA-210{circumflex over ( )}7 HSV-2 49 5% 1.3 H19 HZ-1 NG-7, NG-8 0 42 10% 1.0 H19 HZ-1 NG-7, NG-8 10{circumflex over ( )}4 HSV-1 76 3% 1.8 H19HZ-1 NG-7, NG-8 10{circumflex over ( )}5 HSV-1 471 5% 11.2 H19 HZ-1NG-7, NG-8 10{circumflex over ( )}7 HSV-2 47 9% 1.1

EXAMPLE 9 TSHC Detection Reduces Vector Background

The TSHC assay eliminates the vector contamination problem oftenassociated with the Hybrid Capture II (HC II) detection assay (Digene).As the RNA signal sequence probes used in HC II are generated fromlinearized vector templates, any remaining unlinearized plasmid DNAresults in the production of additional RNA probe sequences specific forvector sequences. In the HC II assay, the RNA/DNA hybrids that form as aresult of these read-through transcripts are captured on the antibodycoated plates and generate signal. In contrast, in the TSHC method, onlythose RNA/DNA hybrids that also hybridize to the capture sequence probesare detected. Accordingly, any detection of vector-related sequences iseliminated. Plasmids SK+, pBR322, DgZ and 1066 which were known to bedetectable in HSV HC II test (Digene) were tested in the TSHC assayusing two RNA signal sequence probes (H19 and RH5b) and two capturesequence probes (VH-2 and VH-4). Identical set of RNA probes were thenused in HC II method and the TSHC method for the detection of HSV-1. Thegeneral TSHC method described in Example 1 was employed. As shown inTable 16, while signal to noise ratio in standard HC II ranged from 14to 48, the signal to noise ratio for the TSHC method was less than 2 forall plasmids tested.

TABLE 16 Vector Background in TSHC v. HCII Detection Method SSP CSPTargets/ml RLU CV S/N TSHC H19 + RH5B VH-2 + VH-4 0 94 6% 1.0 TSHC H19 +RH5B VH-2 + VH-4 4 ng pBS SK+ 137 7% 1.5 TSHC H19 + RH5B VH-2 + VH-4 2ng pBR322 99 6% 1.1 TSHC H19 + RH5B VH-2 + VH-4 4 ng DgX 135 7% 1.4 TSHCH19 + RH5B VH-2 + VH-4 4 ng 1066 107 7% 1.1 HC II H19 + RH5B None 0 949% 1.0 HC II H19 + RH5B None 4 ng pBS SK+ 4498 3% 48.1 HC II H19 + RH5BNone 2 ng pBR322 1281 8% 13.7 HC II H19 + RH5B None 4 ng DgX 2003 5%21.4 HC II H19 + RH5B None 4 ng 1066 1536 2% 16.4

EXAMPLE 10 Sensitivity and Specificity of detecting HSV-1 and HSV-2 byTSHC

The sensitivity and typing discrimination for the TSHC detection ofHSV-1 and HSV-2 were assessed using the TSHC described in Example 1. Inthe HSV-1 TSHC assay, signal sequence probes H19 and RH5B, capturesequence probes HZ-1, VH-2 and VH-4, and blocker probes NG-7, NG-8,GP-3, GP-4, and GP-1 were used. In the HSV-2 TSHC assay, signal sequenceprobes 18 and Ei8, capture sequence probes NF-1 and NF-2, and blockerprobes HX-4, HX-5 and GP-8 were used. HSV-1 and HSV-2 viral particleswere diluted to various concentrations using the Negative ControlSolution. As shown in FIGS. 4 and 5, while 104 copies of the eitherHSV-1 or HSV-2 (450 copies/well) were detected in the respective assays,there was virtually no detection of the cross-reactive type HSV atconcentrations up to and including 10⁸ copies/ml (4,500,000copies/well). Thus, the HSV-1 and HSV-2 TSHC assays can distinguish thetwo HSV types at a greater than 10,000-fold range of discriminationwhile maintaining excellent sensitivity (450 VP/well).

The HSV-1 TSHC assay shows a linear range of detection ranging from atleast 2×10³ to 5×10³ VP/ml (Table 17). The specificity of the assay isexcellent as no cross-reactivity was detected (S/N is less than or equalto 2) in samples containing HSV-2 at a concentration as high as 2×10⁷ to5×10⁷ viral particles/ml. Similarly, the HSV-2 TSHC assay also showsexcellent specificity, wherein no cross-reactivity was detected insamples containing HSV-1 at a concentration as high as 5×10⁷ viralparticles/ml (Table 18). Similar results were obtained from TSHCdetection of HSV-2 using a dilution series of HSV-2 and HSV-1 viruses(Table 19).

TABLE 17 Analytical Sensitivity and Specificity of the HSV1 TSHC AssayTargets RLU S/N Negative Control 47 1.0 HSV2 @ 5 × 10{circumflex over( )}7 VP/ml 57 1.2 HSV2 @ 2 × 10{circumflex over ( )}7 VP/ml 43 0.9 HSV1@ 5 × 10{circumflex over ( )}3 VP/ml 201 4.3 HSV1 @ 2 × 10{circumflexover ( )}3 VP/ml 107 2.3

TABLE 18 Analytical Sensitivity and Specificity of the HSV2 TSHC AssayTargets RLU S/N Negative Control 40 1.0 HSV1 @ 5 × 10{circumflex over( )}7 VP/ml 78 2.0 HSV1 @ 2 × 10{circumflex over ( )}7 VP/ml 55 1.4 HSV2@ 5 × 10{circumflex over ( )}3 VP/ml 218 5.5 HSV2 @ 2 × 10{circumflexover ( )}3 VP/ml 106 2.7

TABLE 19 Detection with HSV-2 Probes using HSV-1 and HSV-2 of DifferentDilution Targets RLU S/N Negative Control 43 1.0 HSV1 @ 5 ×10{circumflex over ( )}7 VP/ml 112 2.6 HSV1 @ 2 × 10{circumflex over( )}7 VP/ml 57 1.3 HSV1 @ 1 × 10{circumflex over ( )}7 VP/ml 38 0.9 HSV1@ 1 × 10{circumflex over ( )}6 VP/ml 38 0.9 HSV1 @ 1 × 10{circumflexover ( )}5 VP/ml 33 0.8 HSV1 @ 1 × 10{circumflex over ( )}4 VP/ml 52 1.2HSV1 @ 1 × 10{circumflex over ( )}3 VP/ml 43 1.0 HSV1 @ 1 ×10{circumflex over ( )}2 VP/ml 39 0.9 HSV2 @ 1 × 10{circumflex over( )}7 VP/ml 257173 5980.8 HSV2 @ 1 × 10{circumflex over ( )}6 VP/ml28544 663.8 HSV2 @ 1 × 10{circumflex over ( )}5 VP/ml 3200 74.4 HSV2 @ 1× 10{circumflex over ( )}4 VP/ml 266 6.2 HSV2 @ 5 × 10{circumflex over( )}3 VP/ml 181 4.2 HSV2 @ 1 × 10{circumflex over ( )}3 VP/ml 62 1.4HSV2 @ 1 × 10{circumflex over ( )}2 VP/ml 44 1.0

EXAMPLE 11 Clinical Specimen Testing

A 64-member clinical specimen panel was tested for HSV-1 and HSV-2 usingboth TSHC and HCII methods. The panel included 15 samples containingknown quantities of HSV-1 or HSV-2, and 49 samples known to be negativefor HSV-1 and HSV-2 by PCR testing. Accordingly, the 15 positive sampleswere “Expected” to test positive in both the HCII and TSHC assays, andthe 49 negative samples were “Expected” to test negative in both theHCII and TSHC tests.

The general TSHC method described in Example 1 was employed. The resultsusing the HCII method and the TSHC method are shown in Tables 20 and 21,respectively. Of the 49 samples “Expected” to yield negative result, 5samples tested positive and 44 samples tested positive using the HCIImethod. In comparison, all 49 samples tested negative using the TSHCmethod. Therefore, the TSHC method is superior in specificity to theHCII method in the detection of HSV-1 and HSV-2.

TABLE 20 Observed vs. Expected Results for HCII Detection of HSV1 andHSV2 Expected Result HCII Result Positive Negative Positive 15 5Negative 0 44 Total 15 49

TABLE 21 Observed vs. Expected Results for TSHC Detection of HSV1 andHSV2 Expected Result TSHC Result Positive Negative Positive 14 0Negative 1 49 Total 15 49

EXAMPLE 12 Effect of Combining Probes in TSHC Detection of HSV

The effect of combining HSV-1 specific signal sequence probe and capturesequence probe sets on HSV-1 detection was assessed. TSHC detection ofHSV-1 and HSV-2 cross-reactivity was performed separately with twodifferent sets of RNA signal sequence probe/biotinylated capturesequence probe combinations (Set #1: H19 plus HZ-1; and Set #2: RH5bplus the TS-1 and TS-2). TSHC was also performed with both RNA signalsequence probe/biotinylated capture sequence probe sets combined toassess the effect of combining the two probe sets on sensitivity andcross-reactivity. The general TSHC method described in Example 1 wasemployed. The results shown in Table 22 clearly demonstrate an additiveeffect of combining the two probe sets for HSV-1 detection with noapparent increase in HSV-2 cross-reactivity.

TABLE 22 Sensitivity is Improved by Combining HSV-1 Specific CSPs andSSPs Capture Sequence Signal Sequence Probes Probes VP/ml RLU CV S/NHZ-1 H19 0 60 3% 1.0 HZ-1 H19 10{circumflex over ( )}5 HSV-1 267 4% 4.5HZ-1 H19 10{circumflex over ( )}6 HSV-1 2316 6% 38.9 HZ-1 H1910{circumflex over ( )}7 HSV2  49 2% 0.8 TS-1, TS-2 RH5B 0 78 6% 1.0TS-1, TS-2 RH5B 10{circumflex over ( )}5 HSV-1 291 6% 3.8 TS-1, TS-2RH5B 10{circumflex over ( )}6 HSV-1 2368 11%  30.6 TS-1, TS-2 RH5B10{circumflex over ( )}7 HSV2  75 11%  1.0 HZ-1, TS-1, TS-2 H19, RH5B 070 12%  1.0 HZ-1, TS-1, TS-2 H19, RH5B 10{circumflex over ( )}5 HSV-1457 10%  6.5 HZ-1, TS-1, TS-2 H19, RH5B 10{circumflex over ( )}6 HSV-14263 1% 60.9 HZ-1, TS-1, TS-2 H19, RH5B 10{circumflex over ( )}7 HSV2 67 6% 1.0

EXAMPLE 13 TSHC Detection of HPV18 and HPV45

The relative sensitivity and specificity of TSHC and HCII detection ofHuman Papillomavirus 18 (HPV18) and Human Papillomavirus 45 (HPV45) wascompared. Previous studies have established HPV45 as the mostcross-reactive HPV type to HPV18, and conversely, HPV18 as the mostcross-reactive HPV type to HPV45. In this study, the ability of the twomethods to detect HPV18 and HPV45 was assessed using HPV18 and HPV45plasmid DNA.

Capture sequence probes (CSPs) for each of the four Human Papillomavirustypes: HPV16, HPV18, HPV31, and HPV45, were designed. The criteria usedfor designing the capture sequence probes were: 1) the CSP hybridizationistes do not overlap with the SSP sites; 2) the CSPs contain sequencesunique to one HPV type with no stretches of sequence homology to otherHPV types greater than 12 bases; and 3) the CSPs are of sufficientlength so as to be capable of hybridizing efficiently at 70° C.

The blocker probes for each CSP were designed such that they could beadded simultaneously with the CSP during hybridization to the targetnucleic acid. The blocker probes have a melting temperature of at least37° C. but no higher than 60° C., as calculated by the Oligo 5.0 program(National Biosciences, Inc., Plymouth, Minn.). Two blocker probes wereused for each capture oligonucleotide to maximize the blocker effectduring the room temperature plate capture step. It was also desired thatthe blocker probes for each CSP have similar melting temperatures.

CSPs for each of the HPV types were tested for relative captureefficiency and cross-reactivity to other HPV types. CSPs that providedthe best combination of sensitivity and low cross-reactivity were usedfor the detection of HPV using TSHC.

In TSHC and HCII detection of HPV18, HPV18 DNA was used at aconcentration of 10 pg/ml. HPV45, used for cross-reactivity testing, wasused at 4 ng/ml. The general TSHC method described in Example 1 wasemployed. As shown in Table 23, a signal to noise ratio of 16.9 wasobtained for TSHC detection of HPV18 compared to a ratio of 7.6 obtainedfor HCII detection of HPV18. On the other hand, cross-reactivity withHPV45 was significantly reduced using the TSHC method (S/N of 1.3 forTSHC compared to S/N of 393.3 for HCII). The results clearly show thatcompared to the HCII method, the TSHC method for the detection of HPV18was superior in both sensitivity and specificity. Results obtained inexperiments comparing TSHC and HCII detection of HPV45 demonstrate thatthe TSHC method for the detection of HPV45 is superior in bothsensitivity and specificity (Table 24).

TABLE 23 TSHC Detection of HPV 18 Method Target SSP CSP S/N TSHC 0 18L118-7L 1.0 HPV18 (10 pg/ml) 18L1 18-7L 16.9 HPV45 (4 ng/ml) 18L1 18-7L1.3 HC II 0 18L1 none 1.0 HPV18 (10 pg/ml) 18L1 none 7.6 HPV45 (4 ng/ml)18L1 none 393.3

TABLE 24 TSHC Detection of HPV 45 Method Target SSP CSP S/N TSHC 0 45L1ON-1 1.0 HPV45 (10 pg/ml) 45L1 ON-1 8.4 HPV18 (4 ng/ml) 45L1 ON-1 1.6 HCII 0 45L1 none 1.0 HPV45 (10 pg/ml) 45L1 none 8.2 HPV18 (4 ng/ml) 45L1none 494.0

EXAMPLE 14 Target-Specific Hybrid Capture-Plus Assay Protocol

Hepatitis B Virus (HBV) was used as the model system for the developmentof the target-specific hybrid capture-plus (TSHC-plus) assay for thedetection of target nucleic acids.

The hybridization in the TSHC-plus method (FIG. 6A-6D) may be performedin a single step. In the one-step method, CSPs, SSPs containingpre-hybridized DNA-RNA duplex, bridge probes (FIG. 6B-6D), and blockerprobes are added simultaneously to the target nucleic acids. Ifhybridization is performed in two steps, CSPs, SSPs withoutpre-hybridized DNA-RNA duplex, bridge probes and blocker probes arefirst hybridized to the target nucleic acid. Oligonucleotide probescomplementary to the single stranded nucleic acid sequence in the SSPare then added to the reaction to form the DNA-RNA duplexes. The hybridsare then detected using anti-RNA/DNA antibody as described in Example 1.

Experiments were carried out to detect HBV using TSHC-plus (Examples15-18). The method shown in FIG. 6A was used. Human hepatitis B virus(HBV adw2) plasmid DNA of known concentration (Digene Corp) was dilutedusing HBV negative Sample Diluent (Digene). Various dilutions were madeand aliquoted into individual tubes. The negative Sample Diluent wasused as a negative control. A half volume of the Denaturation Reagent5100-0431 (Digene) was added to the test samples. Test samples wereincubated at 65° C. for 45 minutes to denature the nucleic acids in thesamples.

Following denaturation of the HBV sample, a hybridization solutioncontaining capture sequence probes (CSPs), blocker probes, signalsequence probe comprising a M13 DNA/M13 RNA duplex and a single-strandedDNA sequence capable of hybridizing to HBV sequences was added to thesamples, and incubated at 65° C. for 1-2 hours. Alternatively, thedenatured samples were incubated for 1 hour with a hybridizationsolution containing capture sequence probes (CSPs), blocker probes andM13 DNA plasmid containing HBV complementary sequences for 1 hour.Following the incubation, M13 RNA was added to the reaction and theincubation was continued for an additional hour at 65° C.

Tubes containing reaction mixtures were cooled at room temperature for 5minutes and aliquots were taken from each tube and transferred toindividual wells of a 96-well streptavidin plate (Digene). The plateswere shaken at 1100 rpms for 1 hour at room temperature. The solutionwas then decanted and the plates were washed four times with SNM washbuffer (Digene). The alkaline-phosphatase anti-RNA/DNA antibody DR-I(Digene) was added to each well and incubated for 30 minutes at roomtemperature. The DR-I (Digene) was then decanted and the plates werewashed four times with SNM wash buffer (Digene). Following removal ofthe residual wash buffer, luminescent substrate (CDP-Star, Tropix Inc.)was added to each well and incubated for 15 minutes at room temperature.Individual wells were read on a plate luminometer to obtain relativelight unit (RLU) signals.

EXAMPLE 15

The following tables describe the various probes tested in theexperiments described in Examples 16-18.

TABLE 25 Capture Sequence Probes for HBV Location Size within ProbeSequence (bp) HBV Strand HBV C1 GCTGGATGTGTCTGCGGCGTTT 28 374-401 SenseTATCAT (SEQ ID NO:152) HBV C2 ACTGTTCAAGCCTCCAAGCTGC 27 1861-1877 SenseGCCTT (SEQ ID NO:153) HBV C3 ATGATAAAACGCCGCAGACACA 32 370-401 Anti-TCCAGCGATA sense (SEQ ID NO:154)

TABLE 26 HBV/M13 Clones from which SSPs are Prepared Insert SizeLocation Clone name Vector Cloning site (bp) within HBV SA1 M13 mp 18Eco RI, Hind III 35 194-228 SA2 M13 mp 18 Eco RI, Hind III 34 249-282SA1a M13 mp 19 Eco RI, Hind III 35 194-228 SA2a M13 mp 19 Eco RI, HindIII 34 249-282 SA4 M13 mp 19 Eco RI, Hind III 87 1521-1607

TABLE 27 HBV Blocker probes CSP to which it Size hybri- Probe Sequence(bp) dizes B1 ATGATAAAACGCCG (SEQ ID NO:155) 14 HBV C1 B2 CAGACACATCCAGC(SEQ ID NO:156) 14 HBV C1 B3 AAGGCACAGCTTG (SEQ ID NO:157) 13 HBV C2 B4GAGGCTTGAACAGT (SEQ ID NO:158) 14 HBV C2 B5 TATCGCTGGATGTGTC (SEQ IDNO:159) 16 HBV C3 B6 TCGGCGTTTTATCATG (SEQ ID NO:160) 16 HBV C3

EXAMPLE 16 Effect of Blocker Probes on TSHC-Plus Detection of HBV

During room temperature capture step, excess SSP (M13 RNA/HBV-M13 DNAduplex) non-specifically hybridizing to the CSP are immobilized onto theplate which results in high background signals. In an attempt to reducebackground signal, blocker probes were employed in TSHC-Plus detectionof HBV. The blocker probes were designed to be much shorter than theCSPs so that they are only capable of hybridizing to the capture probesat temperatures well below the hybridization temperatures used in theassay.

Blocker probe sets consisting of two separate oligonucleotides that arecomplementary to the CSPs were used. The blocker probes were added tothe hybridization mixture in 10-fold excess relative to the CSPs. Sincethe blocker probes are much shorter than the CSPs, they do not hybridizewith CSPs at the target hybridization temperature and therefore do notinterfere with the hybridization of the CSPs to the target nucleicacids. Following the hybridization of CSP and target nucleic acids, thesamples were subjected to a room temperature capture step during whichthe blocker probes hybridize with excess CSPs, thus preventing them fromhybridizing to the SSPs. As shown in Table 28, the use of the blockerprobes in the hybridization reaction greatly reduced the backgroundsignals of the assay.

TABLE 28 Effect of Blocker Probes on HBV Detection Capture Probe Blockerprobe Background Signal (RLU) HBV C1 no 17892 HBV C1 B1, B2 424 HBV C2no 9244 HBV C2 B3, B4 398

EXAMPLE 17 Effect of the Length of SSP on TSHC-Plus Detection of HBV

The effect of the length of the DNA sequence inserted into the M13vector for generating the SSP on TSCH-Plus detection of HBV was studied.A positive control containing 20 pg/ml of HBV plasmid DNA was used. Asshown in Table 29, the use of a longer HBV complementary sequence in theSSP (87 base pairs) resulted in a substantial increase in signal ofdetection. The effect is unlikely due to sub-optimal hybridizationtemperature condition since the Tm of the shorter probes is 15 degreeabove the hybridization temperature. As the M13 RNA-DNA duplex formed inthe SSP may act to partially block the complementary DNA sequence in theprobe from hybridizing to the HBV sequences in the target nucleic acids,longer complementary sequences in the SSP may overcome this block.

TABLE 29 Effect of the Length of the Complementary sequence in the SSPon TSHC-Plus Detection of HBV Size of the HBV Tm of the HBV Hybridi-Target DNA Sequence Target DNA Se- zation Signal SSP in SSP (bp) quencein SSP temperature (RLU) SA1 35 83° C. 65° C. 1741 SA2 34 80° C. 65° C.1857 SA4 87 108° C.  65° C. 7978

EXAMPLE 18 TSHC-Plus and HC II Detection of HBV

The relative sensitivity of TSHC-Plus and HC II (Hybrid Capture II,Digene) detection of HBV was compared. HBV positive standards of threedifferent concentrations were tested in the experiments. As shown inTable 30, the signals obtained using the TSHC-Plus detection method wereapproximately two-fold higher than those obtained using the HC IIdetection method.

TABLE 30 TSHC-Plus and HC II Detection of HBV* Target HBV ConcentrationMethod Control 10 pg/ml 20 pg/ml 100 pg/ml HC II 48 2355 4225 21438 TSHCPlus 285 4856 7978 37689 *Signal measured as relative light unit (RLU)

The above description of various preferred embodiments has beenpresented for purposes of illustration and description. It is notintended to be exhaustive or limiting to the precise forms disclosed.Obvious modifications or variations are possible in light of the aboveteachings. The embodiments discussed were chosen and described toprovide illustrations and its practical application to thereby enableone of ordinary skill in the art to utilize the various embodiments andwith various modifications as are suited to the particular usecontemplated. All such modifications and variations are within thesystem as determined by the appended claims when interpreted inaccordance with the breadth to which they are fairly, legally andequitably entitled.

1. A method of detecting a target nucleic acid comprising: a)hybridizing a single-stranded target nucleic acid to a capture sequenceprobe and a signal sequence probe to form double-stranded hybridsbetween said probes and the target nucleic acid, wherein the capturesequence probe and the signal sequence probe are capable of hybridizingto non-overlapping regions within the target nucleic acid and not beingcapable of hybridizing to each other; b) adding a blocker probe to thehybridization reaction, wherein said blocker probe hybridizes to excessnon-hybridized capture sequence probes; c) capturing the hybrid to forma bound hybrid; and d) detecting the bound hybrid.
 2. A method ofdetecting a target nucleic acid comprising: a) hybridizing asingle-stranded target nucleic acid to an immobilized capture sequenceprobe and a signal sequence probe to form double-stranded hybridsbetween said probes and the target nucleic acid, wherein the capturesequence probe and the signal sequence probe are capable of hybridizingto non-overlapping regions within the target nucleic acid and not beingcapable of hybridizing to each other; b) adding a blocker probe to thehybridization reaction, wherein said blocker probe hybridizes to excessnon-hybridized capture sequence probes; c) detecting the bound hybrid.3. The method of claim 1, wherein the capture sequence probe is modifiedwith at least one ligand.
 4. The method of claim 1, wherein the signalsequence probe is unlabelled.
 5. The method of claim 3, wherein theligand is biotin.
 6. The method of claim 5, wherein the capture sequenceprobe is linear having a 5′ and 3′ end, wherein both the 5′ and the 3′ends are biotinylated.
 7. The method of claim 1, wherein the capturesequence probe and the signal sequence probe hybridize to regions of thetarget nucleic acid, wherein the regions are less than 3 kilobasesapart.
 8. The method of claim 1, wherein the capture sequence probe andthe signal sequence probe hybridize to regions of the target nucleicacid, wherein the regions are less than 500 bases apart.
 9. The methodof claim 1, wherein the capture sequence probe is a fusion of two ormore sequences complementary to different regions of the target nucleicacid or to different target molecules.
 10. The method of claim 1,wherein the double-stranded hybrid formed is a DNA-RNA hybrid.
 11. Themethod of claim 1, further comprising the step of formingsingle-stranded DNA prior to the hybridization step.
 12. The method ofclaim 1, wherein hybridization of the capture sequence probe and thesignal sequence probe to the target nucleic acid are performedsequentially.
 13. The method of claim 1, wherein step a) and step b) areperformed simultaneously.
 14. The method of claim 1, wherein the blockerprobe has lower melting temperature than that of the capture sequenceprobe.
 15. The method of claim 1, wherein the hybrid is captured onto asolid phase.
 16. The method of claim 15, wherein the solid phase iscoated with streptavidin.
 17. The method of claim 15, wherein the solidphase is a microplate.
 18. The method of claim 1, wherein step c) iscarried out at room temperature.
 19. The method of claim 1, wherein thebound hybrid is detected using an antibody capable of recognizing ahybrid.
 20. The method of claim 19, wherein the hybrid is aDNA-RNA-hybrid.
 21. The method of claim 20, wherein the antibody capableof recognizing a DNA-RNA hybrid is labelled with alkaline-phosphatase.22. A method of detecting a target nucleic acid comprising: a)hybridizing a single-stranded target nucleic acid to a capture sequenceprobe and a signal sequence probe, wherein the capture sequence probeand the signal sequence probe are capable of hybridizing tonon-overlapping regions within the target nucleic acid and not beingcapable of hybridizing to each other, wherein said hybridization formsan RNA-DNA hybrid between said signal sequence probe and the targetnucleic acid; and b) detecting the RNA-DNA hybrid by binding an antibodycapable of recognizing the RNA-DNA hybrid to said hybrid, wherein saidantibody is detectably labelled.
 23. The method of claim 22, furthercomprising capturing the hybrid formed in step a) to form a boundhybrid.
 24. The method of claim 22, wherein the capture sequence probeis modified with at least one ligand.
 25. The method of claim 22,wherein the signal sequence probe is unlabelled.
 26. The method of claim24, wherein the capture sequence probe is biotinylated.
 27. The methodof claim 26, wherein the capture sequence probe is linear having a 5′and a 3′ end, wherein both the 5′ and the 3′ ends are biotinylated. 28.The method of claim 22, wherein the capture sequence probe and thesignal sequence probe hybridize to regions of the target nucleic acid,wherein the regions are less than 3 kilobases apart.
 29. The method ofclaim 22, wherein the capture sequence probe and the signal sequenceprobe hybridize to regions of the target nucleic acid, wherein theregions are less than 500 bases apart.
 30. The method of claim 22,further comprising the step of forming single-stranded target DNA priorto the hybridization step.
 31. The method of claim 22, whereinhybridizations of the capture sequence probe and the signal sequenceprobe to the target nucleic acid are performed sequentially.
 32. Themethod of claim 1, wherein the hybrid formed in step a) is captured ontoa solid phase.
 33. The method of claim 30, wherein the capture step iscarried out at room temperature.
 34. The method of claim 22, wherein thesolid phase is coated with streptavidin.
 35. The method of claim 22,wherein the solid phase is a microplate.
 36. The method of claim 22,wherein the antibody is labelled with alkaline-phosphatase.
 37. Themethod of claim 20, further comprising adding a blocker probe to thehybridization step, wherein said blocker probe hybridizes to excessnon-hybridized capture sequence probes.
 38. The method of claim 37,wherein the blocker probes are added to the hybridization reactionfollowing the hybridization of the capture sequence probes to the targetnucleic acid.
 39. The method of claim 37, wherein the blocker probe haslower melting temperature than that of the capture sequence probe.
 40. Amethod of detecting a target nucleic acid comprising: a) hybridizing asingle stranded target nucleic acid to a capture sequence probe and asignal sequence probe, wherein the capture sequence probe and the signalsequence probe are capable of hybridizing to non-overlapping regionswithin the target nucleic acid and not being capable of hybridizing toeach other, wherein the signal sequence probe comprises a DNA-RNA hybridregion, wherein said hybridization forms a complex; and b) detectingsaid complex.
 41. The method of claim 40 wherein the capture sequenceprobe is immobilization on a solid matrix.
 42. The method of claim 40wherein said complex is detected by binding an antibody capable ofrecognizing the DNA-RNA hybrid region to said region, wherein theantibody is detectably labelled.
 43. The method of claim 40 wherein thecapture sequence is modified with at least one ligand.
 44. The method ofclaim 43 wherein the ligand is biotin.
 45. The method of claim 44wherein two biotin molecules are attached to the capture sequence probe.46. The method of claim 40, further comprising adding a blocker probeafter the hybridization step, wherein said blocker probe hybridizes toexcess non-hybridized capture sequence probe.
 47. A nucleic acid probeconsisting of a sequence selected from the group consisting of SEQ IDNO: 1 through SEQ ID NO:
 160. 48. The method according to claim 1,wherein the signal sequence probe comprises a DNA-RNA duplex and asingle stranded nucleic acid sequence which is capable of hybridizing tothe target nucleic acid.
 49. The method according to claim 48, whereinthe DNA-RNA duplex is a M13 DNA-M13 RNA duplex.
 50. A method ofdetecting a target nucleic acid comprising: a) hybridizing asingle-stranded target nucleic acid to a capture sequence probe, abridge probe and a signal sequence probe to form double-stranded hybridsbetween said capture sequence and bridge probes and the target nucleicacid, wherein the capture sequence probe and the bridge probe arecapable of hybridizing to non-overlapping regions within the targetnucleic acid and not being capable of hybridizing to each other, and thesignal sequence probe is capable of hybridizing to the bridge probe andnot being capable of hybridizing to the target nucleic acid and thecapture sequence probe; b) adding a blocker probe to the hybridizationreaction, wherein said blocker probe hybridizes to excess non-hybridizedcapture sequence probes; c) capturing the hybrid to form a bound hybrid;and d) detecting the bound hybrid.
 51. The method according to claim 50,wherein the signal sequence probe comprises a DNA-RNA duplex and asingle stranded nucleic acid which is capable of hybridizing to thebridge probe.
 52. The method according to claim 51, wherein the DNA-RNAduplex is a M13 DNA-M13 RNA duplex.
 53. The method according to claim51, wherein the DNA-RNA duplex is a hybrid formed between repeatsequences within the signal sequence probe and a nucleic acid moleculehaving complementary sequences to the repeat sequences.
 54. The methodaccording to claim 50, wherein the bridge probe further comprises apoly(A) tail.
 55. The method according to claim 54, wherein the signalsequence probe comprises a single stranded poly(dT) DNA sequence whichis capable of hybridizing to the poly(A) tail of the bridge probe, and aDNA-RNA duplex formed between the poly(dT) sequences in the signalsequence probe and a nucleic acid molecule having poly(A) sequences.